The present invention relates generally to microfluidic devices and flow-based methods for performing analytical testing and analysis and, more specifically, the present invention provides compositions and methods for signal enhancement of surface plasmon resonance-based chemical detection in flow-based systems, such as microfluidic flow devices.
Surface plasmon resonance (SPR) is a general spectroscopic method for sensing refractive index changes near the surface of a metal film. Its sensitivity to these changes provides a versatile platform for the observation and quantitation of chemical reactions and intermolecular binding at the metal/solution interface. The generality of the technique has led to its application to a variety of chemical systems, including biological interactions and reactions. Several specifically designed commercial instruments are currently available for these types of assays.
SPR allows detection of small changes in refractive index that result from interactions between surface-bound biomolecules and a solution-borne binding partner. For example, immobilization of a protein to the sensor surface allows for detection of protein binding events manifested by a change in refractive index that is measured as a change in the angle-dependent (or wavelength-dependent) reflectance of the metal film. This type of SPR sensing is typically carried out on commercial instruments that can use, for example, a binding or immobilization layer, such as a carboxylated dextran gel, in addition to a gold film as the sensor surface. Where the dextran gel is used, the gel acts as a host for the surface-immobilized binding partner. However, SPR has also been applied in a number of other formats, including imaging SPR where a large number of chemistries can be rapidly interrogated simultaneously. Also, a variety of other surface chemistries have been used to immobilize and pattern biomolecules for interaction with their binding partners.
SPR relies on the optical excitation of surface modes (plasmons) in a free electron metal, e.g., gold (Au), silver (Ag), aluminum (Al), or copper (Cu) anchored to a glass substrate. Methods for attaching or anchoring the metal to the glass substrate are well known in the art and can include adhesion with a thin layer of, for example, mercaptosilane, titanium, or chromium. Back-side, p-polarized illumination of a prism-coupled film at a specific angle greater than the critical angle for total internal reflection results in plasmon excitation at the metal-solution interface. SPR is most easily observed as a reduction in the intensity of reflected light as measured at the detector (located in the path of the reflected light). The experimental condition (angle or wavelength) of minimum reflectivity, denoted as the SPR angle, shifts to a different position as material is adsorbed onto the metal layer. The shift in the resonance position can be converted to a measure of the thickness of the adsorbed material using various calculations, e.g., complex Fresnel calculations. Adsorption, desorption, and molecule-molecule interactions that occur within the sensing region of about 300 nm adjacent to the metal-solution interface, can thus be monitored in real-time, making SPR suitable for dynamic sensing.
In using SPR to test for biological, biochemical, or chemical substances, a beam of light from a laser source is directed through a prism onto a biosensor consisting of a transparent substrate, usually glass, which has one external surface covered with a thin film of a noble metal, which in turn, is covered with an organic film that interacts strongly with an analyte, such as a biological, biochemical, or chemical substance. The organic film can contain substances, such as antibodies or antigens, that can bind with an analyte in a sample solution to cause an increase in the refractive index in the sample sensing region and shift the SPR resonance. By monitoring either the position of the SPR resonance (as a function of the experimental parameter angle or the wavelength) or the reflectivity at fixed experimental parameters near the SPR resonance, the presence or absence of an analyte in the sample can be detected. Typically, labeling of biomolecules is not required, nor has it been desired. However, this limits the changes in refractive index produced by the binding of both analytes and secondary reagents, which, in turn, limits the speed and sensitivity of SPR detection.
Materials and products have been proposed to enhance the signal from standard SPR-detection methods. One such method is described in WO 01/09388 (incorporated herein by reference), wherein metal nanoparticles are used as optical tags.
The present invention provides additional compositions and methods for signal enhancement of surface plasmon resonance-based chemical detection systems. In the methods an enzyme precipitation signal-enhancement protocol that is well established for use in optical absorption assays is shown to increase the signal associated with reflectivity changes in chemical assays, especially biomolecular recognition assays on planar SPR surfaces coated with a capture reagent. The methods are generally applicable to all detection schemes in which an enzyme label can be used. Commonly used detection schemes in which this method is applicable include, but are not limited to: i) a competition or displacement assay with an enzyme labeled ligand, ii) a sandwich assay in which the enzyme is linked to an agent that binds specifically to the analyte of interest, i.e., an antibody, and iii) a nucleic acid assay in which the enzyme is linked to a nucleic acid probe fragment.